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Fig. 3 Brachyury-GFP degradation by oligoTRAFTACs. (A) Brachyury-targeting oligonucleotide used in the oligoTRAFTAC design engaged with brachyury-GFP. Brachyury targeting biotinylated oligonucleotide (BRCH) or its scrambled oligonucleotide (SCRM) incubated with cell lysate and captured by streptavidin agarose beads. (n = 2) (B) Two oligoTRAFTACs with 3? VHL ligand modifications, OT3 (5 PEG unit linker) and OT4 (2 PEG unit linker) were transfected into HEK293T cells and brachyury-GFP levels were analyzed in lysates prepared after 20 h. (n = 2) (C) OT3 induced brachyury-GFP degradation as early as 12 h in HEK293T cells. (n = 2) (D) Washout experiment after 12 h of OT3 transfection. Cells were incubated continuously for 24 h in transfection medium or OT3 was aspirated after 12 h of transfection and fresh medium added to cells. Washout cells incubated for another 12 h and 24 h prior to harvesting. (n = 2) (E) Chemical structure of brachyury-targeting oligoTRAFTAC (OT3). OT3 consists of a phosphodiester backbone.